Fatty Acid Binding and Conformational Stability of Mutants of Human Muscle Fatty Acid-binding Protein

Overview

Journal Biochem J

Specialty Biochemistry

Date 1996 Feb 15

PMID 8660291

Citations 11

Authors

C F Prinsen

J H Veerkamp

Affiliations

Soon will be listed here.

Abstract

Human muscle fatty acid-binding protein (M-FABP) is a 15 kDa cytosolic protein which may be involved in fatty acid transfer and modulation of non-esterified fatty acid concentration in heart, skeletal muscle, kidney and many other tissues. Crystallographic studies have suggested the importance of the amino acids Thr-40, Arg-106, Arg-126 and Tyr-128 for the hydrogen bonding network of the fatty acid carboxylate group. Two phenylalanines at 16 and 57 are positioned to interact with the acyl chain of the fatty acid. We prepared 13 mutant proteins by site-directed mutagenesis and tested them for fatty acid binding and stability. Substitution of amino acids Phe-16, Arg-106 or Arg-126 created proteins which showed a large decrease in or complete loss of oleic acid binding. Substitution of Phe-57 by Ser or Val and of Tyr-128 by Phe had no great effect. The stability of the mutant proteins was tested by denaturation studies on the basis of fatty acid binding or tryptophan fluorescence and compared with that of the wild-type M-FABP. There was no direct relationship between fatty acid-binding activity and stability. Less stable mutants (F57S and Y128F) did not show a marked change in fatty acid-binding activity. Substitution of Arg-126 by Gln or Arg-106 by Thr eliminated binding activity, but the former mutant protein showed wild-type stability, in contrast to the latter. The results are in agreement with crystallographic data.

Citing Articles

An α-synuclein decoy peptide prevents cytotoxic α-synuclein aggregation caused by fatty acid binding protein 3.

Fukui N, Yamamoto H, Miyabe M, Aoyama Y, Hongo K, Mizobata T J Biol Chem. 2021; 296:100663.

PMID: 33862084 PMC: 8131325. DOI: 10.1016/j.jbc.2021.100663.

A fatty acid-binding protein of facilitates the acquisition of host polyunsaturated fatty acids.

Gullett J, Cuypers M, Frank M, White S, Rock C J Biol Chem. 2019; 294(44):16416-16428.

PMID: 31530637 PMC: 6827280. DOI: 10.1074/jbc.RA119.010659.

Comparative study of the fatty acid binding process of a new FABP from Cherax quadricarinatus by fluorescence intensity, lifetime and anisotropy.

Li J, Henry E, Wang L, Delelis O, Wang H, Simon F PLoS One. 2013; 7(12):e51079.

PMID: 23284658 PMC: 3528769. DOI: 10.1371/journal.pone.0051079.

The human fatty acid-binding protein family: evolutionary divergences and functions.

Smathers R, Petersen D Hum Genomics. 2011; 5(3):170-91.

PMID: 21504868 PMC: 3500171. DOI: 10.1186/1479-7364-5-3-170.

Protein reconstitution and three-dimensional domain swapping: benefits and constraints of covalency.

Carey J, Lindman S, Bauer M, Linse S Protein Sci. 2007; 16(11):2317-33.

PMID: 17962398 PMC: 2211703. DOI: 10.1110/ps.072985007.

References

Sha R, Kane C, Xu Z, BANASZAK L, Bernlohr D . Modulation of ligand binding affinity of the adipocyte lipid-binding protein by selective mutation. Analysis in vitro and in situ. J Biol Chem. 1993; 268(11):7885-92. View

Xu Z, Bernlohr D, BANASZAK L . The adipocyte lipid-binding protein at 1.6-A resolution. Crystal structures of the apoprotein and with bound saturated and unsaturated fatty acids. J Biol Chem. 1993; 268(11):7874-84. View

Scapin G, Young A, Kromminga A, Veerkamp J, Gordon J, Sacchettini J . High resolution X-ray studies of mammalian intestinal and muscle fatty acid-binding proteins provide an opportunity for defining the chemical nature of fatty acid: protein interactions. Mol Cell Biochem. 1993; 123(1-2):3-13. DOI: 10.1007/BF01076469. View

Eads J, Sacchettini J, Kromminga A, Gordon J . Escherichia coli-derived rat intestinal fatty acid binding protein with bound myristate at 1.5 A resolution and I-FABPArg106-->Gln with bound oleate at 1.74 A resolution. J Biol Chem. 1993; 268(35):26375-85. View

Thumser A, Evans C, Worrall A, Wilton D . Effect on ligand binding of arginine mutations in recombinant rat liver fatty acid-binding protein. Biochem J. 1994; 297 ( Pt 1):103-7. PMC: 1137797. DOI: 10.1042/bj2970103. View

Veerkamp J, van Kuppevelt T, Maatman R, Prinsen C . Structural and functional aspects of cytosolic fatty acid-binding proteins. Prostaglandins Leukot Essent Fatty Acids. 1993; 49(6):887-906. DOI: 10.1016/0952-3278(93)90174-u. View

Banaszak L, Winter N, Xu Z, Bernlohr D, Cowan S, Jones T . Lipid-binding proteins: a family of fatty acid and retinoid transport proteins. Adv Protein Chem. 1994; 45:89-151. DOI: 10.1016/s0065-3233(08)60639-7. View

LaLonde J, Bernlohr D, BANASZAK L . X-ray crystallographic structures of adipocyte lipid-binding protein complexed with palmitate and hexadecanesulfonic acid. Properties of cavity binding sites. Biochemistry. 1994; 33(16):4885-95. DOI: 10.1021/bi00182a017. View

Maatman R, van Moerkerk H, Nooren I, van Zoelen E, Veerkamp J . Expression of human liver fatty acid-binding protein in Escherichia coli and comparative analysis of its binding characteristics with muscle fatty acid-binding protein. Biochim Biophys Acta. 1994; 1214(1):1-10. DOI: 10.1016/0005-2760(94)90002-7. View

10.

Young A, Scapin G, Kromminga A, Patel S, Veerkamp J, Sacchettini J . Structural studies on human muscle fatty acid binding protein at 1.4 A resolution: binding interactions with three C18 fatty acids. Structure. 1994; 2(6):523-34. DOI: 10.1016/s0969-2126(00)00052-6. View

11.

Bennett E, Stenvers K, Lund P, Popko B . Cloning and characterization of a cDNA encoding a novel fatty acid binding protein from rat brain. J Neurochem. 1994; 63(5):1616-24. DOI: 10.1046/j.1471-4159.1994.63051616.x. View

12.

LaLonde J, Bernlohr D, BANASZAK L . The up-and-down beta-barrel proteins. FASEB J. 1994; 8(15):1240-7. DOI: 10.1096/fasebj.8.15.8001736. View

13.

De Filippis V, Sander C, Vriend G . Predicting local structural changes that result from point mutations. Protein Eng. 1994; 7(10):1203-8. DOI: 10.1093/protein/7.10.1203. View

14.

Baier L, Sacchettini J, Knowler W, Eads J, Paolisso G, Tataranni P . An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance. J Clin Invest. 1995; 95(3):1281-7. PMC: 441467. DOI: 10.1172/JCI117778. View

15.

Monera O, Kay C, Hodges R . Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions. Protein Sci. 1994; 3(11):1984-91. PMC: 2142645. DOI: 10.1002/pro.5560031110. View

16.

Lassen D, Lucke C, Kveder M, Mesgarzadeh A, Schmidt J, Specht B . Three-dimensional structure of bovine heart fatty-acid-binding protein with bound palmitic acid, determined by multidimensional NMR spectroscopy. Eur J Biochem. 1995; 230(1):266-80. DOI: 10.1111/j.1432-1033.1995.tb20560.x. View

17.

Veerkamp J, Maatman R . Cytoplasmic fatty acid-binding proteins: their structure and genes. Prog Lipid Res. 1995; 34(1):17-52. DOI: 10.1016/0163-7827(94)00005-7. View

18.

LOWRY O, ROSEBROUGH N, FARR A, RANDALL R . Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1):265-75. View

19.

Glatz J, Veerkamp J . A radiochemical procedure for the assay of fatty acid binding by proteins. Anal Biochem. 1983; 132(1):89-95. DOI: 10.1016/0003-2697(83)90429-3. View

20.

Kunkel T . Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985; 82(2):488-92. PMC: 397064. DOI: 10.1073/pnas.82.2.488. View